With the advent of the Human Genome Project and the field of pharmacogenomics, which aims to correlate sequence polymorphisms with variations in drug responses and disease susceptibility, a heightened need for improved nucleic acid sequencing methods has become apparent.
The most commonly used sequencing methods are variants on the “Sanger” or “dideoxy” method, in which the enzymatic, template dependent incorporation of a chain-terminating dideoxynucleotide results in the generation of a collection of nucleic acid fragments each ending with the base carried by that analog. When a set of four such reactions is performed, one for each of the bases G, A, T and C, the electrophoretically-separated fragments will form a “ladder” from which the sequence can be read.
The efforts to map genomic sequence polymorphisms and mutations, particularly single nucleotide polymorphisms (“SNPs”), have spawned new sequencing technologies aimed at obtaining small amounts of sequence information (often single nucleotides) from a large number of nucleic acid samples. The so-called “minisequencing” methods are currently performed using fluorescently labeled dideoxynucleotides that are enzymatically incorporated opposite a SNP site.
Both “classical” sequencing methods and “minisequencing” methods are thus dependent upon chain terminating nucleotide analogs. The nucleotide terminators traditionally used in such methods are the dideoxy nucleotides, which are structurally similar to the “naturally occurring” deoxynucleotides but differ in the glycosyl component. The dideoxy chain terminating nucleotides contain a 2′,3′-dideoxyribofuranosyl moiety.
Several acyclic nucleotides also have been used as chain terminators. Nucleotide analogs lacking the deoxyribofuranosyl moiety have been reported in the literature to function as substrates for viral DNA polymerases (e.g., Holy et al., 1990, Antiviral Res. 13: 295-312; Martinez et al., 1997, Bioorg. Med. Chem. Lett. 7: 3013-3016; Martinez et al., 1999, Nucleic Acids Res. 27: 1271-1274). The acyclic nucleoside phosphonomethoxyadenine (PME-A) has been reported to exhibit therapeutic properties in the treatment of virus-induced diseases (e.g., HSV). PME-A differs in structure from traditional nucleosides in that it has a phosphonomethoxyethyl group in place of the traditional ribofuranosyl moiety. PME-A labeled with 14C by replacement of a 12C atom in the adenine nucleobase was reported by Merta et al., 1990, Neoplasma 37: 111-120. The 14C-labeled PME-A was used to demonstrate that PME-A can be phosphorylated in mouse leukemia cell extracts; detection followed TLC separation of phosphorylated (PME-Ap, PME-App) from non-phosphorylated forms of PME-A.
Phosphonomethoxy carbocyclic nucleosides and nucleotides are taught by Mansuri et al., U.S. Pat. No. 5,744,600. The nucleotide analogs taught therein are said to have use as antiviral agents.
Because of increasing demand for sequencing technologies dependent upon chain terminators, there is a need in the art for alternative chain terminators, particularly for terminators that are less costly than dideoxynucleotides.